Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

海马锥体神经元的钾通道和树突功能

• 批准号: 7594222
• 负责人: Dax A Hoffman
• 金额: $ 75.43万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594222
• 关键词: Action Potentials; Acute; Affect; Alzheimer' s Disease; Architecture; Axon; Back; Biophysics; Brain; Brain region; Cell physiology; Cells; Chemosensitization; Chromosome Pairing; Collaborations; Complement; Computer Simulation; Condition; Data; Dendrites; Dendritic Spines; Dominant-Negative Mutation; Doxycycline; Emotions; Epilepsy; Event; Excision; Exhibits; Family; Fire - disasters; Fluorescence; Frequencies; Gene Expression; Gene Proteins; Genes; Green Fluorescent Proteins; Head; Hippocampus (Brain); Image; Imaging Techniques; Kinetics; Knowledge; Kv4.2 channel; Label; Learning; Life; Maintenance; Measures; Memory; Modification; Molecular; Molecular Cloning; Morphologic artifacts; Mus; Mutation; NR1 gene; Nature; Neuraxis; Neurons; Numbers; Pattern; Phase; Physiological; Potassium Channel; Predisposition; Property; Protein Overexpression; Proteins; Protocols documentation; Recovery; Resistance; Role; Second Messenger Systems; Seizures; Shapes; Signal Transduction; Sindbis Virus; Slice; Synapses; Synaptic plasticity; Synaptophysin; System; Techniques; Tetracycline; Tetracyclines; Time; Trans-Activators; Transgenic Mice; United States National Institutes of Health; Variant; Vertebral column; Virus; Voltage-Gated Potassium Channel; Width; Work; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; amino 3 hydroxy 5 methylisoxazole 4 propionate; density; dentate gyrus; electrical property; experience; hippocampal pyramidal neuron; human NR1 protein; morris water maze; mutant; neuronal cell body; neurophysiology; patch clamp; promoter; research study; response; second messenger; size; trafficking; voltage; voltage clamp; voltage gated channel

In the dendrites of hippocampal CA1 pyramidal neurons, a nonuniform density of subthreshold, rapidly inactivating potassium channels regulates signal propagation. This nonuniform distribution (with higher expression in the dendrites than in the soma) means that the electrical properties of the dendrites are markedly different from those of the soma. Incoming synaptic signals are shaped by the activity of these channels, and action potentials, once initiated in the axon, progressively decrease in amplitude as they propagate back into the dendrites. Combining patch clamp recording with molecular techniques, the Molecular Neurophysiology and Biophysics Unit investigates the electrophysiological properties and molecular nature of the voltage-gated channels expressed in CA1 dendrites, how their expression is regulated, and what their role is in learning and memory. Kv4.2 control of firing patterns in hippocampal CA1 pyramidal neurons. Although recent molecular cloning studies have found several families of voltage-gated K channel genes expressed in the mammalian brain, at present, information regarding the relationship between the protein products of these genes and their various neuronal functions is lacking. Our lab has used a combination of molecular, electrophysiological, imaging techniques to show that the voltage gated potassium channel subunit Kv4.2 controls AP half-width, frequency-dependent AP broadening and dendritic action potential propagation. As Ca2 influx occurs primarily during AP repolarization, Kv4.2 activity can regulate cellular processes involving Ca2-dependent second messenger cascades such as gene expression and synaptic plasticity. We are currently developing techniques which will enable us to directly record from dendrites where we have altered voltage-gated channel functional expression. Kv4.2 trafficking in CA1 pyramidal neuron dendrites. Using a modified Sinbis virus system to overexpress EGFP-labeled Kv4.2 (Kv4.2g) in cultured hippocampal neurons, we found that the EGFP fluorescence in dendritic spines of Kv4.2g expressing neurons appeared brighter than that from the adjacent dendritic shaft. The ratio of spine head to dendritic shaft fluorescence in Kv4.2g expressing neurons was approximately two-fold greater than in neurons expressing EGFP. Kv4.2 expression in spines was further shown using electronmicroscopy in collaboration with Ron Petralia here at the NIH. We found stimulation (AMPA) to result in an activity-dependent redistribution of Kv4.2g away from spines to the dendritic shaft and a punctate accumulation of Kv4.2g within the soma. This AMPA-induced redistribution of Kv4.2g occurred within 15 min of stimulation and was reversible, indicating that the treatment was not excitotoxic (6 h washout). Co-expression with pre- and post-synaptic markers (synaptophysin and NR1) showed that Kv4.2 undergoes activity-induced redistribution without a gross change in synaptic architecture or number. We have confirmed these findings with live imaging of Kv4.2g removal from the spine in response to AMPA stimulation. The large number of synapses stimulated in these conditions enabled us to directly measure the effect of internalization as a decrease in the endogenous whole-cell transient K current from uninfected hippocampal neurons without a change in sustained or non-inactivating delayed rectifier-type voltage-gated K current amplitudes. Thus, activity-dependent Kv4.2 internalization occurs natively and is not an artifact of overexpression. We are currently investigating the requirements and mechanisms of Kv4.2 activity-dependent trafficking. Role of voltage-gated potassium channels in synaptic plasticity. Using the Sindbis virus system to infect organotypic slice cultures with Kv4.2g and Kv4.2g(W362F), we have begun investigating the role of Kv4.2 in LTP using a depolarization pairing protocol. For the first 10 min after pairing, potentiation is similar in all three groups, achieving 100% increase in EPSC size. After this period, however, Kv4.2 overexpressing neurons fail to maintain potentiation such that EPSC size is back to baseline after 25 min. Conversely, expression of Kv4.2g(W362F) results in a potentiation, which reaches a greater level 40-50 min after initiation, compared to controls. These data indicate that Kv4.2 channels modulate the degree of LTP by influencing the induction of a late phase of potentiation or by controlling the mechanisms of LTP maintenance. We are currently characterizing the mechanisms of Kv4.2s effect on LTP. Creation and characterization of Kv4.2 transgenic mice. We are currently characterizing a transgenic mouse expressing a dominant negative pore mutation in the voltage-gated potassium channel subunit Kv4.2. This mouse expresses the mutant Kv4.2 channel along with GFP under control of a tetracycline transactivator (tTA) responsive promoter. Expression is spatially controlled by a new line of tTA expressing mice that limit tTA activity to the CA1 and dentate gyrus regions of the hippocampus. Expression can be controlled temporally by administration of doxycycline. Experiments in acute hippocampal slices from these mice will be used to investigate Kv4.2s role in regulating AP backpropagation into CA1 dendrites and in synaptic integration and plasticity. In collaboration with Dr. Anne Anderson, we are investigating seizure susceptibility in these mice. In addition, we are using these mice to investigate Kv4.2s role in hippocampal dependent learning and memory in the Morris water maze. Role of auxiliary proteins in regulating Kv4.2 expression and function. A-type K currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic events, action potentials and neuronal firing. To achieve this diversity, different neuron types express specific complements of Kv4.2 auxiliary subunits. In hippocampal CA1 pyramidal neurons, DPPX is a prominently expressed subunit, which restores many properties of native CA1 A-type currents when co-expressed with Kv4.2 in heterologous systems. To investigate the physiological role of DPPX in CA1 neurons we developed, in collaboration with Bernardo Rudys lab, short-interfering RNAs (siRNAs) to suppress the expression of all DPPX variants. To investigate whether DPPX alters the kinetics of A-type currents in a native system, we conducted voltage-clamp experiments in outside-out patches from CA1 pyramidal neurons in hippocampal organotypic slices infected with siDPPX using a modified Sindbis virus system. We found that siDPPX results in a delayed recovery from inactivation and rightward shifted the steady-state inactivation and activation curves for A-type currents. To determine the physiological effect of the A-type current kinetic modifications by siDPPX, we carried out current-clamp experiments in siDPPX expressing cells. Compared to negative control, siDPPX-infected neurons exhibited decreased input resistance, delayed time to AP onset, increased AP threshold, increased AP half-width and reduced fast AHP amplitudes. Thus siDPPX had contrasting effects, decreasing excitability subthreshold and increasing excitability suprathreshold. We have used computer modeling to determine which of these sub- and suprathreshold effects can be explained by these shifts.

在海马CA1锥体神经元的树突中，亚阈值的非均匀密度，迅速失活的钾通道可调节信号传播。 这种非均匀分布（在树突中的表达较高，而不是在soma中）意味着树突的电性能明显不同于躯体的电特性。 传入的突触信号是由这些通道的活性和一旦在轴突中启动的动作电位来塑造的，随着它们将其传播回树突时，振幅逐渐降低。 分子神经生理学和生物物理学单元将斑块夹的记录与分子技术相结合，研究了在CA1树突中表达的电压门控通道的电生理特性和分子性质，它们的表达方式如何调节，以及它们在学习和记忆中的作用。 KV4.2在海马CA1锥体神经元中控制发射模式。 尽管最近的分子克隆研究发现，目前缺乏有关这些基因蛋白质产物与其各种神经元功能之间关系的信息，但目前缺乏有关电压门控k通道基因的几个家族。 我们的实验室使用了分子，电生理成像技术的组合，以表明电压门控钾通道亚基KV4.2控制AP的半宽度，频率依赖性AP扩大和树突状作用电位传播。 由于Ca2涌入主要发生在AP复极化期间，因此KV4.2活性可以调节涉及Ca2依赖性第二质体级联的细胞过程，例如基因表达和突触可塑性。 我们目前正在开发技术，使我们能够直接从改变电压门控通道功能表达的树突记录。 Kv4.2在CA1锥体神经元树突中运输。 使用改良的Sinbis病毒系统，在培养的海马神经元中过表达EGFP标记的KV4.2（KV4.2G），我们发现表达神经元的kv4.2g的树突状刺激的EGFP荧光似乎比相邻的Dendritritic shaft haft型较亮。 表达神经元的Kv4.2g中脊柱头部与树突轴荧光的比率大约比表达EGFP的神经元大约大约二倍。 在NIH的Ron Petralia合作，使用电子显微镜在棘中进一步显示了Kv4.2的KV4.2表达。 我们发现刺激（AMPA）导致活性依赖于Kv4.2g的重新分布，从刺到树突状轴，并在SOMA内进行KV4.2G的点状积累。 这种AMPA诱导的KV4.2G的重新分布发生在刺激后的15分钟内，并且是可逆的，表明该治疗不是兴奋的（6小时清洗）。 与突触前和突触后标记的共表达（突触蛋白和NR1）表明，KV4.2在没有突触结构或数字的总变化的情况下进行活动诱导的重新分布。 我们已经通过对AMPA刺激从脊柱中去除KV4.2G的实时成像来证实这些发现。 在这些条件下刺激的大量突触使我们能够直接测量内源性全细胞瞬态K电流的降低，而不会改变未感染的海马神经元，而不会改变持续或无激活的延迟反整流电压电压电压电压k电流的k电流放大器。 因此，活性依赖性kv4.2内在化本地发生，不是过表达的伪像。 我们目前正在研究KV4.2活动依赖性贩运的要求和机制。 电压门控钾通道在突触可塑性中的作用。 使用sindbis病毒系统，用Kv4.2g和Kv4.2g（W362F）感染器官型切片培养物，我们开始使用去极化配对方案研究KV4.2在LTP中的作用。 在配对后的前10分钟内，这三组的增强性相似，EPSC尺寸增加了100％。 但是，在此期间之后，KV4.2过表达神经元无法维持增强，因此EPSC的大小在25分钟后恢复到基线。 相反，与对照组相比，KV4.2G（W362F）的表达会导致增强，在开始后40-50分钟后达到更高的水平。 这些数据表明，KV4.2通道通过影响增强后期的诱导或控制LTP维护机制来调节LTP的程度。我们目前正在表征KV4.2S对LTP的效果的机制。 Kv4.2转基因小鼠的创建和表征。 我们目前正在表征转基因小鼠在电压门控钾通道亚基KV4.2中表达显性负孔突变。 该小鼠在控制四环素反式激活器（TTA）响应启动子的控制下表达突变的KV4.2通道。 表达在空间上由一种新的TTA线表达小鼠，该系列将TTA活性限制在海马的CA1和齿状回区域。 表达可以通过施用强力霉素进行时间控制。 这些小鼠的急性海马切片中的实验将用于研究KV4.2S在调节AP反向传播中的作用，并在突触整合和可塑性中调节AP反向传播。 与安妮·安德森（Anne Anderson）博士合作，我们正在研究这些小鼠的癫痫敏感性。 此外，我们正在使用这些小鼠研究莫里斯水迷宫中海马依赖的学习和记忆中的Kv4.2s的作用。 辅助蛋白在调节KV4.2表达和功能中的作用。 A型K电流具有独特的动力学和电压依赖性特性，使它们可以很好地调节突触事件，动作电位和神经元触发。 为了实现这种多样性，不同的神经元类型表达了KV4.2辅助亚基的特定补充。 在海马CA1锥体神经元中，DPPX是一个突出表达的亚基，当在异源系统中与KV4.2共表达时，它恢复了天然Ca1 A型电流的许多特性。 为了研究DPPX在CA1神经元中的生理作用，我们与Bernardo Rudys Lab合作开发了短暂的RNA（siRNA），以抑制所有DPPX变体的表达。 为了研究DPPX是否改变了天然系统中A型电流的动力学，我们使用改良的Sindbis病毒系统在海马器官型切片中的CA1锥体神经元的外部斑块中进行了电压钳实验。 我们发现SIDPPX导致从失活中延迟恢复，并向右移动了A型电流的稳态失活和激活曲线。 为了确定SIDPPX A型电流动力学修饰的生理效应，我们在SIDPPX表达细胞中进行了电流钳实验。 与阴性对照相比，SIDPPX感染的神经元表现出降低的输入电阻，延迟的AP发作时间，AP阈值增加，AP半宽度增加并减少了快速AHP振幅。 因此，SIDPPX具有对比的效果，降低了兴奋性亚阈值并增加了兴奋性上线。 我们已经使用计算机建模来确定这些转变可以解释的这些子和距离效应中的哪些。